Conventional approaches to vaccine development have implemented either whole replication competent virus which has been attenuated (e.g., Sabin polio vaccine, measles, mumps, rubella (MMR)) or inactivated virions that are not replication competent. On occasions, the inactivated virus vaccines may include split vaccines where the virus particles have been disrupted. Molecular techniques have also been used to develop the subunit vaccine (e.g., hepatitis B vaccine) that consists only of the surface glycoproteins of hepatitis B virus. The inactivated virus vaccines tend to induce primarily antibody (Ab) responses to the viruses in question, whereas the live attenuated vaccines induce both cell-mediated immunity as well as an antibody response since the vaccine induces a transient infection.
The only disease which has been eliminated by virtue of a successful vaccination campaign is smallpox. A campaign is currently in progress to eradicate polio. Features of virus infections that can be eliminated by vaccination are infections caused by viruses with stable virus antigens (i.e., very low mutation frequency, few subtypes), that lack a reservoir in other animal species, viruses that do not persist in the body once the infection is over and where vaccination leads to long lasting immunity. Viruses such as polio and measles fulfill these criteria whereas viruses such as influenza virus (Flu), HCV, and HIV that vary their protein sequences do not. It is for this reason that new and alternate approaches are required.
With regard to HIV, the HIV-1 envelope (Env) complex of glycoproteins gp120 and gp41 is the target for neutralizing antibodies (Abs) induced in HIV-1 infected patients and for HIV/AIDS vaccine development (Wyatt et al., “The HIV-1 Envelope Glycoproteins: Fusogens, Antigens, and Immunogens,” Science 280:1884-1888 (1998); Ward et al., “Insights into the Trimeric HIV-1 Envelope Glycoprotein Structure,” Trends Biochem. Sci. 40:101-107 (2015)). Glycoprotein gp120 has been conventionally divided into five variable and five conserved regions (Modrow et al., “Computer-Assisted Analysis of Envelope Protein Sequences of Seven Human Immunodeficiency Virus Isolates: Prediction Of Antigenic Epitopes in Conserved and Variable Regions,” J. Virol. 61:570-578 (1987)), and the region of the first and second variable loops (V1V2) is the most diverse region of Env in both sequence and length (Zolla-Pazner et al., “Structure-Function Relationships of HIV-1 Envelope Sequence-Variable Regions Refocus Vaccine Design,” Nat. Rev. Immunol. 10:527-535 (2010)). However, recent data have shown that V1V2 can form, in the structurally constrained scaffolded V1V2 or the stabilized BG505 SOSIP.664 trimer, a unique five-stranded β-barrel structure with strands A, B, C, C′, and D (Pancera et al., “Structure and Immune Recognition of Trimeric Pre-Fusion HIV-1 Env,” Nature 514:455-461 (2014); Pan et al., “The V1V2 Region of HIV-1 gp120 Forms a Five-Stranded Beta Barrel,” J. Virol. 89:8003-8010 (2015)). In the trimer context, the V1V2 domain is located at the distal apex of the Env trimer, and the three V1V2 regions in the trimer join together at the apex center to form a top layer of the Env complex (Pancera et al., “Structure and Immune Recognition of Trimeric Pre-Fusion HIV-1 Env,” Nature 514:455-461 (2014); Julien et al., “Crystal Structure of a Soluble Cleaved HIV-1 Envelope Trimer,” Science 342:1477-1483 (2013); Lyumkis et al., “Cryo-EM Structure of a Fully Glycosylated Soluble Cleaved HIV-1 Envelope Trimer,” Science 342:1484-1490 (2013); Lee et al., “Cryo-EM Structure of a Native, Fully Glycosylated, Cleaved HIV-1 Envelope Trimer,” Science 351:1043-1048 (2016)). This layer can shield the co-receptor binding sites as well as partially occlude the third variable region (V3); it can also make large movements upon CD4 receptor binding to expose the co-receptor binding sites (Pancera et al., “Structure and Immune Recognition of Trimeric Pre-Fusion HIV-1 Env,” Nature 514:455-461 (2014); Munro et al., “Conformational Dynamics of Single HIV-1 Envelope Trimers on the Surface of Native Virions,” Science 346:759-763 (2014); Spurrier et al., “Structural Analysis of Human and Macaque Mabs 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 Gp120,” Structure 19:691-699 (2011)). V1V2 also harbors a putative integrin-binding site that may also mediate Env binding to host cells (Arthos et al., “HIV-1 Envelope Protein Binds to and Signals Through Integrin Alpha4beta7, the Gut Mucosal Homing Receptor For Peripheral T Cells,” Nat. Immunol. 9:301-309 (2008); Tassaneetrithep et al., “Cryptic Determinant of alpha4beta7 Binding in the V2 Loop of HIV-1 gp120,” PLoS One 9:e108446 (2014); Peachman et al., “Identification of New Regions in HIV-1 gp120 Variable 2 and 3 Loops That Bind to alpha4beta7 Integrin Receptor,” PLoS One 10:e0143895 (2015)). One such site, the tripeptide LDI/V motif at amino acid positions 179-181 (HxB2 numbering) (Ratner et al., “Complete Nucleotide Sequences of Functional Clones of the AIDS Virus,” AIDS Res Hum. Retroviruses 3:57-69 (1987), which is hereby incorporated by reference in its entirety), is located at the beginning of the C′ strand in the β-barrel (Pan et al., “The V1V2 Region of HIV-1 gp120 Forms a Five-Stranded Beta Barrel,” J. Virol. 89:8003-8010 (2015)).
The spatial location of V1V2 on the surface of the Env spike makes it a natural target for the human immune system. HIV-infected individuals can make cross-reactive V1V2 Abs, and many human anti-V1V2 monoclonal antibodies (mAbs) have been isolated (Moore et al., “Probing the Structure of the V2 Domain of Human Immunodeficiency Virus Type 1 Surface Glycoprotein gp120 With a Panel of Eight Monoclonal Antibodies: Human Immune Response to the V1 and V2 Domains,” J. Virol. 67:6136-6151 (1993); Gorny et al., “Human Anti-V2 Monoclonal Antibody That Neutralizes Primary But Not Laboratory Isolates of Human Immunodeficiency Virus Type 1,” J. Virol. 68:8312-8320 (1994); Gorny et al. “Identification of a New Quaternary Neutralizing Epitope on Human Immunodeficiency Virus Type 1 Virus Particles,” J. Virol. 79:5232-5237 (2005); Walker et al., “Broad and Potent Neutralizing Antibodies from an African Donor Reveal a New HIV-1 Vaccine Target,” Science 326:285-289 (2009); Bonsignori et al., “Analysis of a Clonal Lineage of HIV-1 Envelope V2/V3 Conformational Epitope-Specific Broadly Neutralizing Antibodies and Their Inferred Unmutated Common Ancestors,” J. Virol. 85:9998-10009 (2011); Doria-Rose et al., “Developmental Pathway For Potent V1V2-Directed HIV-Neutralizing Antibodies,” Nature 509:55-62 (2014); Israel et al., “Prevalence of a V2 Epitope in Clade B Primary Isolates and its Recognition by Sera from HIV-1-Infected Individuals,” AIDS 11:128-130 (1997); Liao et al., “Vaccine Induction of Antibodies Against a Structurally Heterogeneous Site of Immune Pressure Within HIV-1 Envelope Protein Variable Regions 1 and 2,” Immunity 38:176-186 (2013)). Epitopes for some of these mAbs have been characterized, and were recently classified into three major types—V2i, V2p, and V2q (Mayr et al., “Epitope Mapping of Conformational V2-Specific Anti-HIV Human Monoclonal Antibodies Reveals an Immunodominant Site in V2,” PLoS One 8:e70859 (2013); Spurrier et al., “Functional Implications of the Binding Mode of a Human Conformation-Dependent V2 Monoclonal Antibody Against HIV,” J. Virol. 88:4100-4112 (2014)). The V2i type is defined by a panel of human mAbs, including 830A, 697-D, and 2158 (Gorny et al., “Human Anti-V2 Monoclonal Antibody That Neutralizes Primary But Not Laboratory Isolates of Human Immunodeficiency Virus Type 1,” J. Virol. 68:8312-8320 (1994); Mayr et al., “Epitope Mapping of Conformational V2-Specific Anti-HIV Human Monoclonal Antibodies Reveals an Immunodominant Site in V2,” PLoS One 8:e70859 (2013); Spurrier et al., “Functional Implications of the Binding Mode of a Human Conformation-Dependent V2 Monoclonal Antibody Against HIV,” J. Virol. 88:4100-4112 (2014); Gorny et al., “Functional and Immunochemical Cross-Reactivity of V2-Specific Monoclonal Antibodies From HIV-1-Infected Individuals,” Virology doi:10.1016/j.virol.2012.02.003 (2012); Nyambi et al., “Conserved and Exposed Epitopes on Intact, Native, Primary Human Immunodeficiency Virus Type 1 Virions of Group M,” J Virol 74:7096-7107 (2000); Pinter et al., “The V1/V2 Domain of Gp120 is a Global Regulator of the Sensitivity of Primary Human Immunodeficiency Virus Type 1 Isolates to Neutralization by Antibodies Commonly Induced Upon Infection,” J. Virol. 78:5205-5215 (2004)). Extensive immunological, mutagenesis and structural data have shown that the V2i epitopes overlap with the LDI/V integrin-binding site, and Abs of this family recognize discontinuous regions in V1V2 (Pan et al., “The V1V2 Region of HIV-1 gp120 Forms a Five-Stranded Beta Barrel,” J. Virol. 89:8003-8010 (2015); Mayr et al., “Epitope Mapping of Conformational V2-Specific Anti-HIV Human Monoclonal Antibodies Reveals an Immunodominant Site in V2,” PLoS One 8:e70859 (2013); Spurrier et al., “Functional Implications of the Binding Mode of a Human Conformation-Dependent V2 Monoclonal Antibody Against HIV,” J. Virol. 88:4100-4112 (2014); Gorny et al., “Functional and Immunochemical Cross-Reactivity of V2-Specific Monoclonal Antibodies From HIV-1-Infected Individuals,” Virology 427(2):198-207 (2012)). The V2p type is defined by human mAbs CH58 and CH59 isolated from a vaccinee of the Phase III RV144 human vaccine trial (Liao et al., “Vaccine Induction of Antibodies Against a Structurally Heterogeneous Site of Immune Pressure Within HIV-1 Envelope Protein Variable Regions 1 And 2,” Immunity 38:176-186 (2013); Rerks-Ngarm et al., “Vaccination with ALVAC and AIDSVAX to Prevent HIV-1 Infection in Thailand,” N. Engl. J. Med. 361:2209-2220 (2009); Bonsignori et al. “Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies From an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family,” J. Virol. 86:1152111532 (2012)). Monoclonal Abs CH58 and CH59 react with V2 peptides, indicating that the V2p epitopes are structurally unconstrained and have a helical or helical-coil structure (Liao et al., “Vaccine Induction of Antibodies Against a Structurally Heterogeneous Site of Immune Pressure Within HIV-1 Envelope Protein Variable Regions 1 And 2,” Immunity 38:176-186 (2013)). The V2q type was defined as a quaternary neutralizing epitope and is represented by mAb, including human mAbs PG9 and PG16 (Walker et al., “Broad and Potent Neutralizing Antibodies From an African Donor Reveal a New HIV-1 Vaccine Target,” Science 326:285-289 (2009)). Crystal structures of PG9 and PG16 in complex with engineered V1V2 scaffolds have shown that these mAbs recognize a region in strand C of V1V2, via a strand-strand interaction, as well as two N-linked glycans using the head of the long CDR H3 region harbored by these V2q mAbs (McLellan et al., “Structure of HIV-1 gp120 V1/V2 Domain With Broadly Neutralizing Antibody PG9,” Nature 480:336-343 (2011); Pancera et al., “Structural Basis For Diverse N-Glycan Recognition by HIV-1-Neutralizing V1-V2-Directed Antibody PG16,” Nat. Struct. Mol. Biol. 20:804-813 (2013)). In the V1V2-scaffolds used to crystallize these mAbs, the V1V2 (from ZM109 or CAP45) is grafted into a β-hairpin region in the protein G B1 domain (PDB ID 1FD6) so that the V1V2 is structurally constrained to maintain the conformation found in the trimeric apex (Pancera et al., “Structure and Immune Recognition of Trimeric Pre-Fusion HIV-1 Env,” Nature 514:455-461 (2014)). Although the protein region of the V2q epitopes overlaps that of the V2p epitopes, the structure for former have a β-strand conformation while that for the latter a helical conformation. The V2p mAbs have weak and very restricted neutralizing activity (Liao et al., “Vaccine Induction of Antibodies against a Structurally Heterogeneous Site of Immune Pressure within HIV-1 Envelope Protein Variable Regions 1 And 2,” Immunity 38:176-186 (2013)), while the V2q-specific mAbs PG9 and PG16 have been shown to neutralize greater than 70% of virus strains tested (Walker et al., “Broad and Potent Neutralizing Antibodies from an African Donor Reveal a New HIV-1 Vaccine Target,” Science 326:285-289 (2009)). Thus, the V2q epitope region can be a major site of vulnerable on Env; designing immunogens that can induce Ab responses that target this conformation has not yet been accomplished.
Data from the correlates analysis of the human clinical vaccine trial RV144, the only human vaccine trial with moderate but significant efficacy against HIV-1 acquisition, have demonstrated that vaccine-induced IgG Abs targeting V1V2 inversely correlated with the risk of infection (Haynes et al., “Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial,” The New England Journal of Medicine 366:1275-1286 (2012)), and immunologic data delineated the specificity and cross-reactivity of these Abs (Zolla-Pazner et al., “Vaccine-Induced IgG Antibodies to V1V2 Regions of Multiple HIV-1 Subtypes Correlate with Decreased Risk of HIV-1 Infection,” PLoS One 9:e87572 (2014); Zolla-Pazner et al., “Analysis of V2 Antibody Responses Induced in Vaccinees in the ALVAC/AIDSVAX HIV-1 Vaccine Efficacy Trial,” PLoS One 8:e53629 (2013)). The Abs induced by the vaccine reacted with a V1V2-MuLV gp70 fusion protein, a reagent recognized by the V2i Abs (Gorny et al., “Functional and Immunochemical Cross-Reactivity of V2-Specific Monoclonal Antibodies From HIV-1-Infected Individuals,” Virology doi:10.1016/j.virol.2012.02.003 (2012)). There was no evidence of the elicitation of V2q Abs by the RV144 vaccine in that little neutralizing activity was detected, and neutralization was not associated with reduced infection rates (Haynes et al., “Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial,” The New England Journal of Medicine 366:1275-1286 (2012); Montefiori et al., “Magnitude and Breadth of the Neutralizing Antibody Response in The RV144 and Vax003 HIV-1 Vaccine Efficacy Trials,” J. Infect. Dis. 206:431-441 (2012)). A sieve analysis that compared the V1V2 sequences of viruses from placebo and those from vaccine recipients identified two positions of immune pressure on the virus in V2, residues 169 and 181, supporting the hypothesis that V1V2 Abs correlated with the reduced risk of infection (Rolland et al., “Increased HIV-1 Vaccine Efficacy Against Viruses with Genetic Signatures in Env V2,” Nature 490:417-420 (2012)). These findings suggest that V1V2 can serve as an important target for HIV vaccine development, but the modest efficacy of RV144 indicates the need for a more efficacious vaccine.
Non-neutralizing antibodies (Abs) can protect against various viral infections, contributing to protection from alphaviruses, flaviviruses, respiratory syncytial virus, and cytomegalovirus, among others (reviewed in Excler et al., “Nonneutralizing Functional Antibodies: A New ‘Old’ Paradigm for HIV Vaccines,” Clin. Vaccine Immunol. 21:1023-1036 (2014); Schmaljohn, A., “Protective Antiviral Antibodies That Lack Neutralizing Activity,” Current HIV Res. 11:345-353 (2013)). While the specificity and affinity of non-neutralizing Abs are dependent on the Fab fragment of Abs to target virions and infected cells, many biologic activities of these Abs are a function of the Fc fragment. Such activities include Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), Ab-dependent cell-mediated virus inhibition (ADCVI), complement activation and fixation, degranulation, and the release of pro-inflammatory cytokines (Holl et al., “Antibody-Mediated Fcgamma Receptor-Based Mechanisms of HIV Inhibition: Recent Findings and New Vaccination Strategies,” Viruses 1:1265-1294 (2009)). Specific examples include protection from herpes simplex 2 in mice by non-neutralizing Abs which mediate ADCC (Balachandran et al., “Protection Against Lethal Challenge of BALB/c Mice by Passive Transfer of Monoclonal Antibodies to Five Glycoproteins of Herpes Simplex Virus Type 2,” Infect. and Immunity 37:1132-1137 (1982); Gorander et al., “Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection After Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis,” Viruses 6:4358-4372 (2014); Petro et al., “Herpes Simplex Type 2 Virus Deleted in Glycoprotein D Protects Against Vaginal, Skin and Neural Disease,” Elife 4:e06054 (2015)), and protection from influenza in mice by non-neutralizing Abs targeting the head or stalk of the influenza hemagglutinin (DiLillo et al., “Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Require FcgammaR Interactions for Protection Against Influenza Virus in Vivo,” Nat. Med. 20:143-151 (2014); Henry et al., “Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection,” Cell Host Microbe 19:800-813 (2016)).
The present invention is directed to overcoming these and other deficiencies in the art.